1. Field of the Invention
The present invention is broadly concerned with an improved process for the accurate spectrophotometric determination of dissolved carbon dioxide (CO.sub.2) in a sample, such as blood, plasma or serum. More particularly, it is concerned with such a process making use of the reaction between phosphoenolpyruvate (PEP) and bicarbonate ion (HCO.sub.3.sup.-) as catalyzed by phosphoenolpyruvate carboxylase (PEPC) to produce oxaloacetate (OA), followed by the indirect determination of the rate of production of OA. A nontoxic enzyme inhibitor is added which renders the kinetics of the system essentially first order over the entire HCO.sub.3.sup.- concentration range of interest. Best results have been obtained when the inhibitor provides a ClO.sub.3.sup.-, ClO.sub.4.sup.-, SCN.sup.-, or I.sup.- anion.
2. Description of the Prior Art
Analysis of body fluids such as blood serum for total dissolved CO.sub.2 content is a widely employed test, useful in determining the acid-base status of a patient. It is also used to monitor patients in renal failure or acute acidosis. Generally speaking, the bicarbonate ion concentration in blood serum or plasma is from about 22 to 32 mmol/L, and under abnormal conditions may range between about 15 to 40 mmol/L. Accordingly, accurate dissolved CO.sub.2 determinations of unknown samples within these concentration ranges is a matter of concern.
In general, prior CO.sub.2 determinations have included manometric, pH indicator, spectrophotometric, and pH related electrode techniques, all of which require acidification of the sample. In addition, enzymatic spectrophotometric methods have been described in which the various forms of CO.sub.2 in the sample are converted to HCO.sub.3.sup.- under alkaline conditions; thereupon, the HCO.sub.3.sup.- is converted to OA, the latter being indirectly measured by consumption of the reduced form of nicotinamide adenine dinucleotide (NADH) and quantitated spectrophotometrically.
In more detail, the enzymatic CO.sub.2 determination described in the prior art makes use of the following coupled reactions: ##STR1## where PEPC is phosphoenolpyruvate carboxylase, P.sub.i is inorganic phosphate anion, OA is oxaloacetate, MDH is malate dehydrogenase and NAD.sup.+ is the oxidized form of NADH. See, e.g., U.S. Pat. No. 3,974,037, which is incorporated by reference herein. As explained in that patent, the bicarbonate reacts with PEP in the presence of PEPC to quantitatively produce OA, whereupon the OA is measured as a function of conversion of NADH to the oxidized form thereof. NADH absorbs UV light between 320-400 nm, with an absorbance peak at 340 nm; NAD.sup.+ on the other hand, has a negligible absorbance at these wave lengths. Accordingly, the conversion of NADH to the oxidized form thereof provides a convenient way to indirectly and quantitatively measure OA.
The foregoing coupled reactions can be used to determine CO2 concentration by allowing the reactions to go to completion and measuring a total change in absorbance. This is referred to as an end point technique, but is not favored because it requires a large reagent-to-sample volume ratio to measure bicarbonate over a wide range of concentrations, and moreover it requires analyzers with high absorbance reading capabilities (low stray light and spectrophotometric noise). In spite of these factors, the end point determination is widely used because until now, a practical wide range kinetic method has not been available.
Alternatively, the CO.sub.2 content of a sample may be determined as a function of how rapidly the enzymatic reaction converts the substrates to products. This rate determination or kinetic method depends on the principle that the rate of the reaction will be proportional to the concentration of a substrate converted by the reaction, where the concentration of the substrate limits the rate of the reaction. The basis of this principle is that, in an enzymatic reaction, the rate at which a substrate is converted to product depends both upon the basic physical rate of the enzyme catalyzed reaction and the frequency at which enzyme molecules and substrate molecules collide. The rate of collision is proportional to the concentration of substrate molecules. As substrate concentration is increased, frequency of collision reaches a maximum and is no longer a factor in determining reaction rate. At this substrate concentration, the concentration of substrate does not limit the rate at which the reaction can enzymatically proceed; only enzyme activity determines the rate of conversion of substrate molecules to product molecules.
Thus, in a rate determination of the content of a substrate in a sample, all other substrates participating in the reaction must be present at concentrations which do not limit the reaction rate, so that, insofar as substrates are concerned, only the concentration of the tested substrate is "rate-limiting". Accordingly, in the use of reaction [I] to kinetically determine HCO.sub.3.sup.- in a sample, only the HCO.sub.3.sup.- should be rate-limiting; PEP must be provided in amounts which do not limit the rate at which PEPC catalyzes the carboxylation of PEP to OA. Where reaction [I] is linked to reaction [II], NADH also must be provided in "non-rate-limiting amounts" and activity units of MDH must be sufficiently greater than the activity units of the PEPC employed so as to catalyze the rate of reaction [II] at a speed no slower than the rate of reaction [I]. Then, since the rate of formation of the OA limits the rate at which NADH is converted to NAD, and since the rate of formation of OA is in turn dependent upon the concentration of HCO.sub.3.sup.- in the sample, spectrophotometric measurement of the NADH absorbance-decrease within a fixed time interval (i.e., the rate of absorbance change) can be used to determine the HCO.sub.3.sup.- concentration in the sample.
In order to correlate a determined rate of absorbance change to the concentration of the substrate at which that rate occurs, it must be known whether the rate of absorbance change is directly proportional, or proportional in a more complex way, to the substrate concentration. This may be learned by repetitively conducting the same test, using the same reagents and under the same conditions, and making the rate of absorbance change measurements at the same fixed time interval, but varying the known concentrations of the substrate. These results are then plotted on a graph of substrate (X-axis) versus change in absorbance (Y-axis). If the relationship between the rate of absorbance change for the fixed time interval and the substrate concentration is linear between a selected zero point and the maximum substrate concentration desired to be determined, then only the rate of absorbance change for one known substrate concentration need be known in order to determine an unknown substrate concentration for which a rate of absorbance change is obtained under the same conditions and same fixed time interval. But if the plotted relationship between the rate of absorbance change and substrate concentration is not linear, resort must be made to the curve described-by such relationship in order to determine an unknown substrate concentration corresponding to a measured rate of absorbance change. Such a standard curve is employed in methods exemplified by that described in the aforementioned U.S. Pat. No. 3,974,037. Stated otherwise, in such prior methods, the relationship between the rate of NADH absorption change and HCO.sub.3.sup.- concentrations is not linear for all HCO.sub.3.sup.- concentrations.
It is known that the rate equation for an enzymatic reaction may be expressed as: ##EQU2## where v=rate of reaction, V=maximal velocity, [S]=substrate concentration, and K.sub.m =the Michaelis-Menten constant. In the case of a true first order reaction, K.sub.m is much greater than [S], so that the latter term becomes negligible. However, in the system of interest, the K.sub.m and [S] values are of the same order of magnitude over the concentration range to be determined. As a consequence, the resulting absorbance plot is curvilinear.
In order to overcome this difficulty, it has been suggested in the past to employ a competitive inhibitor in the reaction system which has the effect of rendering the rate of the reaction linear or directly proportional to all concentrations of HCO.sub.3.sup.- which could be found in a given body sample. For example, European Patent Specification 076,478 published Jan. 15, 1986 (this Specification being incorporated by reference herein) describes the use of inorganic pyrophosphates as inhibitors. In an inhibited system, it is known that the rate equation can be expressed as: ##EQU3## where v, V, [S] and K.sub.m are defined as set forth above, [I]=inhibitor concentration, K.sub.i =inhibitor constant, and K.sub.m (1+[I]/K.sub.i =apparent K.sub.m. As can be appreciated from a study of equation [IV], a suitable inhibitor should be selected to make apparent K.sub.m much greater than [S], so that the resulting rate plot is linear.
While the noted European Patent Specification advocates the use of pyrophosphates as an appropriate inhibitor, others have found it difficult to replicate the results therein reported. Accordingly, there remains an unsatisfied need in the art for an inhibitor which will render the change in absorbance essentially linear over the [HCO.sub.3 ].sup.- concentration range of interest.